Calpain-1 Signaling Pathways in Platelets

描述（由申请人提供）：钙蛋白酶是半胱氨酸蛋白酶，参与多种细胞过程，如细胞骨架组织、细胞增殖、细胞凋亡、细胞运动和止血。我们提出了以下具体目标来检验钙蛋白酶-1通过调节PTP 1B的活性来调节血小板信号传导的假设：（1）钙蛋白酶-1对PTP 1B的调节。我们将使用结构域特异性抗体和免疫复合物磷酸酶测定法确定野生型和无钙蛋白酶-1小鼠血小板活化后PTP 1B裂解的状态。我们将测量PTP 1B的降解和降解片段的比活性，并研究PTP 1B对血小板中c-Src活化的影响。这些研究将有助于阐明钙蛋白酶-1在PTP 1B调节中的作用，并为钙蛋白酶-1缺失小鼠血小板中酪氨酸磷酸酶活性增加提供解释。(2)钙蛋白酶-1依赖性β-3整合素与血小板细胞骨架相互作用的机制。我们计划通过凝胶电泳和质谱法确定血小板中PTP 1B底物的身份。使用β-3整合素胞质结构域的结构进行保守和激进的取代酪氨酸在747和759，我们将确定其磷酸化的化学计量，敏感性去磷酸化的PTP 1B，并量化它们的相互作用与Shc，肌球蛋白，和新发现的PTP 1B基板。在BIAcore技术的辅助下，这些实验将有助于阐明PTP 1B的下游效应物，这些效应物对于血小板中的由外向内信号传导至关重要。(3)钙蛋白酶-1信号在血小板铺展中的作用。使用具有纯C57 BL/6 J遗传背景的calpain-1敲除小鼠，我们将通过鉴定小鼠血小板中calpain-1的潜在PTP 1B非依赖性底物来研究增强血小板铺展的机制。我们还将测试具有纯C57 BL/6 J遗传背景的钙蛋白酶-1缺失小鼠是否显示血小板分泌和体内血栓形成状态的异常。总之，这些研究将有助于阐明钙蛋白酶-1在整合血小板信号传导的共同和不同方面的作用，揭示PTP 1B调控的新方面，并提供对血小板聚集和凝块收缩途径机制的更好理解。最终，这些研究可能会导致新的治疗策略的发展对血栓形成。公共卫生相关性：钙蛋白酶是半胱氨酸蛋白酶，参与多种细胞过程，如细胞增殖、凋亡、细胞运动和止血。这些研究将阐明钙蛋白酶-1在血小板信号传导中的作用，并为更好地理解血小板聚集和凝块收缩途径的机制提供帮助。最终，这些研究可能会导致新的治疗策略的发展对血栓形成。

DESCRIPTION (provided by applicant): Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. We propose to test the hypothesis that calpain-1 regulates platelet signaling by modulating the activity of PTP1B under the following specific aims: (1) Regulation of PTP1B by calpain-1. We will determine the status of PTP1B cleavage in the wild type and calpain-1 null mouse platelets upon activation using domain-specific antibodies and immune complex phosphatase assays. We will measure the degradation of PTP1B and specific activity of degraded fragments and investigate the effect of PTP1B on c-Src activation in platelets. These studies will help to clarify the role of calpain-1 in the regulation of PTP1B and provide an explanation for the increased tyrosine phosphatase activity in calpain-I null mouse platelets. (2) Mechanism of calpain-1 dependent beta-3 integrin interactions with the platelet cytoskeleton. We plan to determine the identity of PTP1B substrates in platelets by gel electrophoresis and mass spectrometry. Using beta-3 integrin cytoplasmic domain constructs carrying conservative and radical replacement of tyrosines at 747 and 759, we will determine their phosphorylation stoichiometry, susceptibility to dephosphorylation by PTP1B, and quantify their interactions with Shc, myosin, and newly identified PTP1B substrates. These experiments, assisted by the BIAcore technology, will help to clarify the downstream effectors of PTP1B that are critical for outside-in signaling in platelets. (3) Role of calpain-1 signaling in platelet spreading. Using calpain-1 null mice with pure C57BL/6J genetic background, we will investigate the mechanism of enhanced platelet spreading by identifying the potential PTP1B-independent substrates of calpain-1 in mouse platelets. We will also test whether the calpain-1 null mice with the pure C57BL/6J genetic background display abnormalities in the platelet secretion and in vivo thrombosis status. Together, these studies will help to clarify the role of calpain-1 in integrating common and divergent aspects of platelet signaling, reveal novel aspects of PTP1B regulation, and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis. PUBLIC HEALTH RELEVANCE: Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cell proliferation, apoptosis, cell motility, and hemostasis. The proposed studies will clarify the role of calpain-1 in platelet signaling and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis.